PROJECT SUMMARY/ABSTRACT Immunoconjugates are designed to focus the delivery of highly cytotoxic agents to specific target cells using monoclonal antibodies, potentially improving the therapeutic index of the agent. To this end, we propose to deliver a cytotoxic protein payload with an anti-EMP2 IgG1 antibody. EMP2, or epithelial membrane protein-2, is a biomarker that is highly expressed in the majority (63%; n=97) of invasive breast cancer tumors examined compared to healthy mammary epithelium, and high EMP2 expression is observed in over 75% of triple negative breast cancer cases examined where EMP2 is observed in both the primary tumor as well as in metastatic lesions. As engineered recombinant antibodies hold great promise for cancer diagnostics and therapy, we have carefully assembled a research team centered at UCLA and The University of Texas MD Anderson Cancer Center to develop recombinant anti-EMP2 antibodies fused to the human serine protease granzyme B for therapy. The group at UCLA has previously shown that a full length IgG1 against EMP2 promotes apoptosis both in vitro and in vivo in a number of EMP2 positive tumors, but as new data suggests that the anti-EMP2 IgG1 can internalize rapidly, we hypothesize that it may serve as a novel candidate for drug conjugation. The use of Granzyme B (GrB) as an immunoconjugate has been proposed as a ?magic bullet? as, once delivered to the cytoplasm of a cell, it activates apoptosis pathways with little to no induction of immunogenicity. The Mohamedali lab has utilized human GrB as an effective payload for the generation of recombinant cell death-inducing fusion proteins and has clearly demonstrated that GrB-containing fusion constructs have impressive and highly selective cytotoxic effects when delivered to the cytoplasm by either antibody or growth factor cell targeting carriers. In this proposal, with the explicit goal of advancing the science of anti-EMP2 antibodies as well as GrB as a cytotoxic payload for clinical testing, we will create and determine the efficacy of two anti-EMP2 IgG1- GrB conjugates. Accordingly, the specific aims propose to design and test the efficacy of GrB linked to an anti-EMP2 IgG1 for cytotoxicity as well as start creating a preclinical package to understand its functional affinity and stability. As the naked antibody cross-reacts between human and mouse, the toxicity profile of the immunoconjugate, including pharmacokinetics and maximum tolerated dosage, will also be determined. Given its high expression in a number of gynecological tumors including ovarian and endometrial, these studies will be important to position EMP2 as a viable target for cancers in women. It will also, given the need for new toxins with low immunogenicity, help position granzyme B in the forefront of antibody linked toxins.